AChE is the target of organophosphate (OP) and carbamate (CB) pesticides, and mutations in the gene can significantly reduce insects' sensitivity to these pesticides. Bombyx mori is highly sensitive to pesticides. To investigate the effects of mutations on AChE1 structure and function, we used a prokaryotic system to express B.mori wild type AChE1 (wAChE1) and mutant AChE1 (mAChE1) in this study. Active AChE1 proteins were obtained after refolding and purification, and wAChE1 and mAChE1 had similar activities. After incubation with 10(-6)M physostigmine and 10(-3)mg/mL phoxim, the remaining enzyme activity of mAChE1 was 4.42% and 8.86% higher than that of wAChE1's, respectively. Three-dimensional analysis of mutation AChE1 (mAChE1) revealed that the Ser and Ala side chains extended toward the central part of S285 with distances of just 2.80A and 3.68A, respectively, which changed the spatial structure of the active center and reduced its sensitivity to pesticides. These results indicated that the mutations altered the 3D structure of AChE1, which may affect the binding of physostigmine and phoxim to the serine residue at the active center, leading to reduced sensitivity. Our study helps understand the relationship between AChE1 mutations and pesticide resistance and provides a new direction for the cultivation of new pesticide-resistant varieties of B.mori.
The main mechanism of toxicity of organophosphate (OP) and carbamate (CB) insecticides is their irreversible binding and inhibition of acetylcholinestrase (AChE), encoded by ace1 (acetylcholinestrase gene 1), leading to eventual death of insects. Mutations in AChE may significantly reduce insects susceptibility to these pesticides. Bombyx mori is an important beneficial insect, and no OP- or CB-resistant strains have been generated. In this study, wild-type ace1 (wace1) and mutant ace1 (mace1) were introduced into BmN cells, confirmed by screening and identification. The expression of wace1 and mace1 in the cells was confirmed by Western blot and their expression levels were about 21-fold higher than the endogenous ace1 level. The activities of AChE in wace1 and mace1 transgenic cells were 10.6 and 20.2% higher compared to control cells, respectively. mace1 transgenic cells had higher remaining activity than wace1 transgenic cells under the treatment of physostigmine (a reversible cholinesterase inhibitor) and phoxim (an OP acaricide). The results showed that ace1 transgene can significantly improve ace1 expression, and ace1 mutation at a specific site can reduce the sensitivity to AChE inhibitors. Our study provides a new direction for the exploration of the relationship between AChE mutations and drug resistance.
The acetylcholinesterase of Lepidoptera insects is encoded by two genes, ace1 and ace2. The expression of the ace1 gene is significantly higher than that of the ace2 gene, and mutations in ace1 are one of the major reasons for pesticide resistance in insects. In order to investigate the effects of the mutations in ace1's characteristic sites on pesticide resistance, we generated mutations for three amino acids using site-directed mutagenesis, which were Ala(GCG)303Ser(TCG), Gly(GGA)329Ala(GCA) and Leu (TCT)554Ser(TTC). The Baculovirus expression system was used for the eukaryotic expression of the wild type ace1 (wace1) and the mutant ace1 (mace1). SDS-PAGE and Western blotting were used to detect the targeting proteins with expected sizeof about 76 kDa. The expression products were purified for the determination of AChE activity and the inhibitory effects of physostigmine and phoxim. We observed no significant differences in the overall activity of the wild type and mutant AChEs. However, with 10 min of physostigmine (10 muM) inhibition, the remaining activity of the wild type AChE was significantly lower than that of the mutant AChE. Ten min inhibition with 33.4 muM phoxim also resulted in significantly lower remaining activity of the wild type AChE than that of the mutant AChE. These results indicated that mutations for the three amino acids reduced the sensitivity of AChE to physostigmine and phoxim, which laid the foundation for future in vivo studies on AChE's roles in pesticide resistance.
Title: Transcriptional characteristics of gene expression in the midgut of domestic silkworms (Bombyx mori) exposed to phoxim Gu ZY, Sun SS, Wang YH, Wang BB, Xie Y, Ma L, Wang JM, Shen WD, Li B Ref: Pesticide Biochemistry and Physiology, 105:36, 2013 : PubMed
Silkworm (Bombyx mori) is not only an economically important insect but also a model system for lepidoptera. As a vital organ of digestion and nutrient absorption, the midgut of insects also serves as the first physiological barrier to chemical pesticides. In this study, microarray was performed to profile the gene expression changes in the midgut of silkworms exposed to phoxim. After 24h of phoxim exposure (4.0mug/mL), 266 genes displayed at least 2.0-fold changes in expression levels. Among them, 192 genes were up-regulated, and 74 genes were down-regulated. The most significant changes were 14.88-fold up-regulation and 23.36-fold down-regulation. According to gene ontology annotation and pathway analysis, differentially expressed genes were mainly classified into different groups based on their potential involvements in detoxification, immunne response, stress response, energy metabolism and transport. Particularly, the transcription levels of detoxification-related genes were up-regulated, such as cytochrome P450s, esterases and glutathione-S-transferase (GST), indicating increased detoxification activity in the midgut. Our study provides new insights into the molecular mechanism of pesticide metabolism in the midgut of insects, which may promote the development of highly efficient insecticides.
Title: Changes in the activity and the expression of detoxification enzymes in silkworms (Bombyx mori) after phoxim feeding Wang YH, Gu ZY, Wang JM, Sun SS, Wang BB, Jin YQ, Shen WD, Li B Ref: Pesticide Biochemistry and Physiology, 105:13, 2013 : PubMed
Silkworm (Bombyx mori) is an economically important insect. However, non-cocoon caused by chemical insecticide poisoning has largely hindered the development of sericulture. To explore the roles of detoxification enzymes in B. mori after insecticide poisoning, we monitored the activity changes of cytochrome P450 monooxygenase, glutathione-S-transferase, and carboxylesterase in B. mori midgut and fatbody after phoxim feeding. At the same time, the expression levels of detoxification enzyme-related genes were also determined by real-time quantitative PCR. Compare to the control levels, the activity of P450 in the midgut and fatbody was increased to 1.72 and 6.72 folds; the activity of GST was no change in midgut, and in fatbody increased to 1.11 folds; the activity of carboxylesterase in the midgut was decreased to 0.69 folds, and in fatbody increased to 1.13 folds. Correspondingly, the expression levels of detoxifying enzyme genes CYP6ae22, CYP9a21, GSTo1 and Bmcce were increased to 15.99, 3.32, 1.86 and 2.30 folds in the midgut and to 3.58, 1.84, 2.14 and 4.21 folds in the fatbody after phoxim treatment. These results demonstrated the important roles of detoxification enzymes in phoxim metabolism. In addition, the detected activities of such enzymes were generally lower than those in cotton bollworms (Helicoverpa armigera), which may contribute to the high susceptibility of B. mori to insecticides. Our findings laid the foundation for further investigations of the molecular mechanisms of organophosphorus pesticide metabolism in B. mori.
Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing approximately 94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.
